Academic journal article Genetics

Plasmid Copy Number Underlies Adaptive Mutability in Bacteria

Academic journal article Genetics

Plasmid Copy Number Underlies Adaptive Mutability in Bacteria

Article excerpt

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KNOWING the source of mutants that arise under selec- tive conditions is critical to understanding evolution and therefore the origins of cancer and the progress of infectious disease. A genetic system to address this problem was de- signed by Cairns and Foster (Cairns et al. 1988; Cairns and Foster 1991). In this system, a population of bacterial tester cells carries a leaky lac frameshift mutation on a low-copy F'Zac plasmid. Following pregrowth under permissive con- ditions in liquid medium, 10® of these cells are plated on selective medium containing lactose as the sole carbon source. Over 1 week, 100 Lac+ revertant colonies accumulate above the nongrowing plated population. These colonies are of two types. One includes stable Lac+ mutant cells whose mutant lac allele has been corrected by a compensating frameshift muta- tion (Foster and Trimarchi 1994; Rosenberg et al 1994). The other includes cells with multiple tandem copies of the orig- inal leaky mutant allele (Andersson et al. 1998). Two general models have been proposed to explain the origin of these colonies. The evidence presented below suggests some prob- lems with both models.

Stress-induced mutagenesis in nongrowing cells

The first explanations of the Cairns system proposed that Lac+ revertants are generated by an evolved mechanism that senses growth cessation (stress) and creates mutations in hopes of genetically resolving the physiological limitation (Cairns et al. 1988; Cairns and Foster 1991; Torkelson et al. 1997). The reversion rate of the Zac mutation during non- selective growth is 10~® per cell per division. The population of 10® cells plated on lactose does not grow but gives rise to ~100 Lac+ revertant colonies over 6 days. The number of revertants could be explained if the 10® plated cells experi- enced a 100-fold increase in genome-wide frameshift muta- tion rate (assuming one division per week). This simple explanation was ruled out because nonrevertant starved cells in the lawn show very little evidence of chromosomal mutagenesis (Foster 1994; Torkelson et al. 1997). The absence of new mutants in the lawn has been explained in two ways-"directed mutation" and "hypermutable states."

The term directed mutation suggests that induced muta- genesis is focused preferentially on the lac region (Foster and Cairns 1992). This could explain why so few mutations are seen in the genome at large. Direction of mutations to func- tionally relevant targets is mechanistically difficult to imagine (Stahl 1988), but it seems clear that the number of Lac+ revertants under selection increases much more than the number of associated mutations (Torkelson et al. 1997). Furthermore, most of the recovered Lac+ revertants show little or no general mutagenesis (Rosche and Foster 1999). It seems likely that if any mutagenesis occurs in this system, it is somehow focused preferentially on the F'Zac plasmid, which carries the lac mutation that is under selection (Foster 1997).

The term hypermutable states suggests that starvation causes an increase in the genome-wide mutation rate but that affects only ~1 in 1000 of the starved population (10s of the plated 10® cells) (Hall 1990; Torkelson et al. 1997). Focusing general mutagenesis on a few cells (instead of a small genomic region) can also explain why unselected mutants were hard to detect in the starved population as a whole. If all lac revertants were caused by genome-wide mutagenesis of a small subset of the population, the general rate of frameshiit mutations (which correct Zac) in the chosen cells would need to increase 10s-fold over that seen in growing cells. That is, ~I00 lac reversion events would have to occur in the mutagenized sub- population (10s cells) in one division-a rate of ICR3 per cell per division. This rate would be a 10s-fold increase over that measured during nonselective growth (ICR8 per cell per di- vision). Increasing the genome-wide mutation rate 10s-fold seems impossibly cosdy in associated lethal mutations (Roth et al. …

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